Characterization of the AcrIIC1 anti‒CRISPR protein for Cas9‒based genome engineering in E. coli

Anti-CRISPR proteins (Acrs) block the activity of CRISPR-associated (Cas) proteins, either by inhibiting DNA interference or by preventing crRNA loading and complex formation. Although the main use of Acrs in genome engineering applications is to lower the cleavage activity of Cas proteins, they can also be instrumental for various other CRISPR-based applications. Here, we explore the genome editing potential of the thermoactive type II-C Cas9 variants from Geobacillus thermodenitrificans T12 (ThermoCas9) and Geobacillus stearothermophilus (GeoCas9) in Escherichia coli. We then demonstrate that the AcrIIC1 protein from Neisseria meningitidis robustly inhibits their DNA cleavage activity, but not their DNA binding capacity. Finally, we exploit these AcrIIC1:Cas9 complexes for gene silencing and base-editing, developing Acr base-editing tools. With these tools we pave the way for future engineering applications in mesophilic and thermophilic bacteria combining the activities of Acr and CRISPR-Cas proteins.


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Reviewer #1 (Remarks to the Author): In their manuscript, "In vivo characterization of the AcrIIC1 anti-CRISPR for Cas9-based genome engineering," Trasanidou and co-authors used AcrIIC1 to selectively inhibit DNA cleavage by thermostable type II-C Cas9 variants (ThermoCas9 and GeoCas9) in E. coli cells and exploit this property for CRISPRi as well as simultaneous base editing and clone selection.
Throughout this study, the authors employed E. coli strains with a genomically integrated GFP to characterize ThermoCas9 and GeoCas9.They found that both Cas9 orthologues were able to induce targeted double-strand breaks, as evidenced by cell/colony depletion.Depletion efficiency was thereby dependent on the protospacer/PAM sequence used.Next, the authors used AcrIIC1 to selectively inhibit Cas9 nuclease activity with the goal of effectively converting Cas9 into a deadCas9.Importantly, however, the efficacy of CRISPRi, as measured by inhibition of GFP expression upon CRISPR effector targeting, was rather low in samples co-expressing Cas9 and AcrIIC1 compared to conventional dCas9-mediated CRISPR.Subsequently, the authors created dCas9-AID fusions for Geoand ThermoCas9 and used them for C:G to T:A conversion in various sequences targeted in the GFP gene.Interestingly, the authors found that the base editing window is particularly long for both dCas9-AID variants, spanning up to 22 base pairs.Finally, the authors constructed a second set of Cas9-AID fusions, this time based on nuclease-competent Cas9s.They then co-expressed AcrIIC1 to greatly reduce, though probably not completely prevent, Cas9 nuclease activity.This resulted in improved "purity" of base editing outcomes, i.e. "clean" point mutations in protospacers amplified from single colonies, and also narrowed the base editing window to PAM-proximal sites.
Overall, the manuscript is well-written and the data are convincing and, for the most part, support the claims made.However, I feel that this paper lacks sufficient novelty to warrant publication in Nature Communications.I acknowledge that there are several interesting data sets in this MS, such as the report of wide base editing windows with the GeoCas9-and ThermoCas9-AIDs.On the other hand, the central claim of the MS is the characterization of AcrIIC1 and its utility for genome engineering.
We would like to thank Reviewer #1 for their useful comments and suggestions.In our study, we demonstrate that the thermostable type II-C ThermoCas9 and GeoCas9 can be used to broaden the targeting scope of genome editing, gene silencing, and base-editing in mesophiles by recognizing alternative PAMs and creating up to six times larger base-editing windows compared to previously reported SpyCas9 base-editors.Complementing this type II-C CRISPR-Cas toolbox, we show that AcrIIC1 can be used not only as an 'off-switch' for genome editing applications (like most anti-CRISPR proteins) but also as a mediator of gene silencing and base-editing in E. coli.To the best of our knowledge, AcrIIC1 has never been characterized in vivo for gene silencing and base-editing purposes.In addition, this is the first report to date on anti-CRISPR-mediated inhibition of the ThermoCas9 activity.Hence, the novelty of our work derives from the exploitation of both the AcrIIC1 and the ThermoCas9/GeoCas9 proteins.
Regarding the first aspect, the mechanism by which AcrIIC1 inhibits type II-C Cas9s, i.e. targeting the HNH domain to selectively prevent Cas9 nuclease function but not DNA binding, has been known for many years (Pawluk et al., Cell 167, 2016).It is not surprising, in my opinion, that this mechanism holds true when the protein is expressed in E. coli.Pawluk et al. (Cell 167, 2016) and more recent studies (Harrington et al., Cell 170, 2017;Song et al., Cell reports 29, 2019;Garcia et al., Cell reports, 2019;Mathony et al., Nature Chemical Biology 16, 2020) showed that AcrIIC1 blocks in vitro the DNA cleavage but not the DNA binding ability of several II-C and some II-A Cas9 endonucleases.However, in vitro functionality of a protein does not guarantee its efficient in vivo activity.In this study, we demonstrated that AcrIIC1 efficiently inhibits the cleavage activity of ThermoCas9 and GeoCas9 in E. coli and we developed a versatile regulatory tool for in vivo CRISPR-based applications (genome editing, gene silencing and base-editing).Notably, AcrIIC1 has never been used for gene silencing and base-editing purposes.
Furthermore, the authors claim to report "the first Acr tools for base-editing applications [...]".This is not true, as, for-instance, AcrIIA5 has already been used to prevent off-target editing in the context of base editing in mammalian cells (Liang et al., Cells 9, 2020; doi:10.3390/cells9081786).
Liang et al. (Cell 9, 2020) applied AcrIIA5 for inhibition of base-editing at undesired loci of the mammalian genome.In contrast, we describe the exploitation of AcrIIC1 for the enhancement of baseediting outcomes in bacteria, for example by providing higher number of clean point mutations.For clarity, we rephrased 'the first Acr tools for base-editing applications [...]' into 'the first Acr tools for the facilitation of base-editing applications […]'.
Moreover, AcrIIC1 seems to be of little use in context of CRISPRi in my opinion.The data in the MS shows that dCas9 is superior for CRISPRi compared to AcrIIC1-Cas9 co-expression, the latter of which is also more complicated from an application perspective because it involves an additional component (AcrIIC1).In addition, if CRISPRi is employed for gene regulation, one would usually want to avoid any risk of unintended DSB induction and would therefore prefer to use well-established dCas9 systems.
The active Cas9:AcrIIC1 chimera would be more desirable than the traditional dCas9 system in gene circuits that require easy alternation between cleavage and binding activity.In the case of the Cas9: AcrIIC1 chimera, this would be achieved by simply inducing or not inducing the expression of AcrIIC1, while the conventional CRISPRi system would necessitate an additional active cas9 gene as well as strictly control regulation of both dCas9 and Cas9.In our study, we provide examples of such gene circuits by developing Acr base-editors that couple base-editing applications (by allowing AcrIIC1 expression) to subsequent counter-selection (by interrupting AcrIIC1 expression).The applicability of these circuits could be further expanded for the study of microbial communities, which are typically composed of non-model organisms, providing an easy and flexible way to control population dynamics.Transient expression of AcrIIC1 could be used to control the growth rate and/or productivity of certain community members, without the requirement for use of a tight expression system.Controlled reduction in the AcrIIC1 expression would eliminate these members from the community, allowing to study the effect of the population changes to the community.For clarity, we added this example in the Discussion section ('In addition, the applicability of our active Cas9:Acr systems for gene targeting inhibition, gene silencing and base-editing could be further expanded for the study of microbial communities, which are typically composed of non-model organisms, providing an easy and flexible way to control population dynamics.Transient expression of AcrIIC1 could be used to control the growth rate and/or productivity of certain community members, without the requirement for use of a tight expression system.Controlled reduction in the AcrIIC1 expression would eliminate these members from the community, allowing to study the effect of the population changes to the community.').
Beyond these critics regarding novelty and applicability of the presented CRISPRi approach, I do acknowledge the utility AcrIIC1 in context of base editing showcased by the authors.My understanding of this system is that AcrIIC1 here serves as a somewhat leaky inhibitor of Cas9 DNA cleavage.Hence, when base editing occurs sufficiently quick and close to the PAM, the resulting cells are protected from cell killing via Cas9-induced DSBs.This is, indeed, a nice strategy taking advantage of the AcrIIC1 inhibitory mechanism and the Acr's "leakiness" regarding Cas9 inhibition.
On the experimental side, the study is very much focused on genomically-integrated GFP as a target.The study would certainly benefit from characterization of their system at endogenous sites, e.g. in the context of the interesting base-editing work the authors have done.I also invite the authors to think about applications that are only possible with their AcrIIC1-based approaches, e.g. in the context of base editing, which have not been possible before.An experimental demonstration of such a biological application would greatly strengthen this paper.Also, the paper would be considerably strengthened, if the authors could showcase their AcrIIC1 base editing strategy in another organism, in particular in mammalian cells.
A major strength of our anti-CRISPR/Cas base-editing tool is the use of the 'leaky' counter-selection nuclease (i.e.Cas9 inhibited by the anti-CRISPR) that kills non-edited cells, as acknowledged by the reviewer.We feel we indeed haven't emphasized this aspect enough in our study.As such, we pointed this out more clearly in the Results section 'AcrIIC1:Cas9 complexes as alternatives of dThermocas9 and dGeocas9 for base-editing in E. coli'' by adding the sentence 'Hence, these Acr base-editors would first induce base-editing of the target site by expressing AcrIIC1 that blocks the Cas9 nuclease activity of the Cas9-PmCDA1 fusion protein and allows for PmCDA1-mediated deamination of the target base(s).Subsequent interruption of the AcrIIC1 expression would result in Cas9-mediated counterselection of the wild-type cells.'.Furthermore, the added value of our Acr base editors is that they offer higher editing purity (higher number of clean mutants) and a higher frequency of mutagenesis (number of nucleotides that are simultaneously mutated) compared to the 'canonical' dCas9 base editors.Taken together, we hope to have shown that our tool offers substantial benefits over current technologies.
Furthermore, in the revised version of our manuscript, we have applied both the dCas9 and the Acr base-editors not only for the genomically-integrated GFP but also for three different endogenous sites (pyrE, xylB and adhE genes).These additional results have been integrated into the manuscript as Supplementary Figures 11 and 12.Moreover, the Results section 'AcrIIC1:Cas9 complexes as alternatives of dThermocas9 and dGeocas9 for base-editing in E. coli', the Methods section 'Baseediting assays' and the Supplementary Tables 2, 3 and 4 have been adjusted accordingly.
Finally, our base-editors are not directly translatable to mammalian cells, due to the difference in repair pathways preferences between prokaryotes and human cells.Especially the prevalence of NHEJ (non-homologous end-joining) in human cells will diminish the benefits that our 'leaky' counterselection approach will have over more conventional approaches.Contrary to base-editing applications in eukaryotes that predominantly use nickase Cas9 variants, our base-editors are dedicated to applications in prokaryotes.
Finally, the word "in vivo" in the title of the paper is somewhat imprecise, since in the context of CRISPR it often refers to use in model organisms such as mice.I am aware that different communities use the term "in vivo" differently, but I would still suggest specifying in the title that the characterization was done in E. coli.
We agree with the reviewer that this might be confusing to some readers.Hence, the title 'In vivo characterization of the AcrIIC1 anti-CRISPR protein for Cas9-based genome engineering' was replaced by 'Characterization of the AcrIIC1 anti-CRISPR protein for Cas9-based genome engineering in E. coli'.
I hope the authors find my comments useful and can understand my reasoning for not supporting publication in Nature Communications.That being said, I think this is an interesting study overall and certainly a good candidate for publication in a more specialized journal.